3. Results

3.1. Molecular Changes in Osteoblasts Co-Cultured with Prostate Cancer Cells
To understand how factors secreted by prostate cancer cells regulate gene expression in osteoblasts we co-cultured rat osteosarcoma derived osteoblastic cells (UMR-106) with an osteolytic prostate cancer cell line (PC3) on transwell plates without physical contact (Figure 1A). UMR cells were also cultured alone as the control. By comparing gene expression in osteoblast co-cultures to monocultures we identified 113 up- and 63 down-regulated genes (Figure 1B, Table S2). Differentially expressed genes included several known regulators of bone metabolism, such as Gpnmb [20], Fhl2 [21], Mgp [22], Enpp1 [23] and Phex [24].
Figure 1  Co-culture of UMR-106 osteoblastic cells with prostate cancer cells (PC3) promotes changes in gene expression. (A) UMR-106 cells were cultured alone or with PC3 cells in transwells, and gene expression changes were quantified using microarrays; (B) 113 and 63 genes were found differentially expressed between UMR cells co-cultured with PC3 cells compared to UMR cells alone; (C) a subset of these differentially transcribed genes were confirmed using qPCR; and (D) immunocytochemistry showed a reduction in Sost protein expression in UMR cells co-cultured with PC3 cells (UMR + PC3) compared to UMR cells cultured alone (UMR alone).   Factors secreted by osteoblasts have been shown to play a major role in regulating cancer metastasis to bone [25,26]. This prompted us to investigate the changes in the regulation of the osteoblast secretome in response to osteoblast-prostate cancer interactions. Our analysis identified 41 genes encoding secreted proteins (Table 1) that are more than two-fold up- or down-regulated in osteoblast co-cultures compared to monocultures. We also found Wnt pathway inhibitors Sost [27] and its paralog Sostdc1 [28] with altered expression in co-cultured osteoblasts. Sost expression was significantly down-regulated, while its paralog Sostdc1 was significantly up-regulated in co-cultured osteoblasts (Table 1). Using qPCR we confirmed the differential expression of a subset of genes, including Mmp13, Ctgf, Il-6, Adamts1, Sost and Sostdc1 (Figure 1C), in osteoblast co-cultures.
microarrays-04-00503-t001_Table 1 Table 1  Genes encoding secreted proteins more than two-fold up- or down-regulated in UMR-106 osteoblastic cells co-cultured with PC3 cells compared to UMR cells cultured alone.

3.2. Functional Analysis of Differentially Regulated Genes
A gene ontology (GO) analysis of differentially expressed genes was performed using ToppGene Suite [16]; “response to hormone”, “extracellular matrix organization”, “cell migration”, “ossification” and “vasculature development” were identified to be a few of the most enriched biological processes. The top 200 enriched biological processes are listed in Table S3, while most relevant GO terms and associated genes are listed in Table 2. Of all the differentially expressed genes, secreted signaling proteins Tgfb2, Il-6, Cxcl1, and Ctgf and metallopeptidases Mmp13, Adamts4, and Adamts5 were of particular interest, as these genes have previously been shown to play a role in regulating cancer migration and invasion [29,30,31,32,33,34,35,36] and bone remodeling [37,38,39,40,41].
microarrays-04-00503-t002_Table 2 Table 2  Enriched gene ontology terms associated with genes differentially expressed between UMR cells co-cultured with PC3 cells and UMR monocultures.   To better understand how secreted signaling proteins Tgfb2, Il-6, Cxcl1, and Ctgf may be involved in potentially regulating a complex biological process such as bone metastasis, we examined these genes in the context of protein interaction networks. GeneMANIA [17] and Cytoscape [18] were used to generate and visualize protein interactions between these genes and related genes in the network. This interaction data included physical and predicted protein–protein interactions. An integrated network of Tgfb2, Il-6, Cxcl1, and Ctgf interactions revealed several putative binding partners in osteoblasts and/or prostate cancer and suggested that these secreted cytokines may control up-regulated transcription factors Junb, Cebpb and Stat3 in osteoblasts (Figure 2). These transcription factors have been shown to play a major role in regulating bone metabolism [42,43,44,45]. This analysis also revealed several other up-regulated genes such as A2m and Nfkbiz as members of Il-6, Cxcl1, Ctgf, and Tgfb2 interactome (Figure 2). Future experimental studies may validate the potential role up-regulation of Il-6, Cxcl1, Ctgf, and Tgfb2 may have in promoting cancer cell migration, invasion, and cancer-induced bone metabolism.
Figure 2  An integrated network of Tgfb2, Il-6, Cxcl1, and Ctgf interactions generated using GeneMANIA and Cytoscape. Query genes (triangles) and up-regulated genes (diamonds) are highlighted.   Metallopeptidases Mmp13, Adamts4, and Adamts5 are associated with the enriched gene ontology term “extra cellular matrix organization”. These metallopeptidases regulate bone remodeling by degrading components of the extracellular matrix, particularly the collagens and aggrecans [36,41,46]. We found Mmp13 to increase greater than 60 fold in osteoblast co-cultures compared to monocultures (Figure 1C). Mmp13 has also been shown to regulate cancer-induced osteolysis [47,48,49]. Adamts4 and Adamts5 have been shown to promote cell growth and invasion [36]. We also identified another up-regulated Adam family member, Adamts1, in osteoblast co-cultures and confirmed the differential regulation of this transcript by qPCR (Figure 1C). Up-regulation of these metallopeptidases may enhance bone remodeling and promote prostate cancer invasion and growth [36,48].

3.3. Effect of Bone Microenvironment Derived Sost on PC3 Gene Expression
Microarray data showed that the Wnt pathway inhibitor Sost was down-regulated in osteoblast co-cultures compared to osteoblast monoculture. We confirmed this observation using qPCR and found a ~3.5-fold reduction in Sost expression in osteoblast co-cultures compared to monocultures (Figure 1C). We also evaluated changes in Sost protein expression using immunocytochemistry and found a significant reduction in Sost expression in UMR cells co-cultured with PC3 cells compared to UMR cells cultured alone (Figure 1D). Wnt signaling has been shown to play a major role in regulating bone metabolism and loss of function of Sost leads to increased bone formation [27]. Disregulated Wnt signaling has also been shown to play a major role in cancer progression and metastasis [50,51,52,53]. This led us to hypothesize that elevated Wnt signaling in the cancer microenvironment due to reduced Sost expression may have a significant effect on prostate cancer metastasis. To understand how Sost levels in the bone microenvironment effect prostate cancer gene expression we co-cultured prostate cancer cells with primary osteoblasts purified from WT and SostKO calvaria and measured gene expression changes in the PC3 cells (Figure 3A).
Figure 3  Co-culture with WT and SostKO osteoblasts (OB) elicited different transcriptional changes in PC3 cells. (A) OBs were isolated and co-cultured with PC3 cells in transwells. 48 h post-plating RNA was isolated and analyzed using microarrays; (B) Heat map showing genes up-regulated in both PC3-WT osteoblast (PC3.WT.OB) co-cultures compared to PC3 alone and PC3-SostKO osteoblast (PC3.SostKO.OB) co-cultures compared to PC3-WT osteoblasts. Individual samples are represented as columns and genes as rows. Four genes from this list, MALAT1, CLCN5, MLL3 and SLC25A36 (in blue rectangle), were found to be up-regulated in metastatic prostate cancer compared to clinically localized cancer.   Our results showed 88 genes up- and one gene down-regulated in response to altered Sost expression (Table S4). Of all the significantly up-regulated transcripts, probes corresponding to 44 transcripts were elevated greater than 1.5-fold in PC3-WT osteoblast co-cultures compared to PC3 monocultures (Figure 3B). Gene ontology analysis of differentially expressed genes revealed that lack of Sost in the bone microenvironment up-regulated several genes associated with the GO term “chemotaxis” including MET, PDGFA, FER, NRP2, and EZR in prostate cancer. Other enriched GO terms include: “cellular protein complex disassembly”, “actin filament organization”, “regulation of cell differentiation”, and “protein phosphorylation” (Table 3).
microarrays-04-00503-t003_Table 3 Table 3  Enriched gene ontology terms associated with genes differentially expressed between PC3-SostKO osteoblast co-cultures and PC3-WT osteoblast co-cultures.   Next, we downloaded metastatic and primary prostate cancer gene expression data [54] from GEO (GSE3325) and identified genes differentially expressed between metastatic prostate cancer and localized primary prostate cancer. Subsequently, we compared genes differentially expressed between PC3 alone, PC3-WT osteoblast co-cultures, and PC3-SostKO osteoblast co-cultures to genes differentially expressed between primary prostate cancer and metastatic prostate cancer. Four genes in particular, MALAT1, CLCN5, MLL3, and SLC25A36, that were up-regulated in metastatic prostate cancer compared to primary prostate cancer were also displayed an enhanced expression in PC3-WT osteoblast co-cultures and PC3-SostKO osteoblast co-cultures compared to PC3 alone (Figure 3B).
MALAT1 is an lncRNA previously shown to promote proliferation and invasion of many cancers, including prostate cancer [55,56]. We found MALAT1 ~4 fold up-regulated in PC3 cells co-cultured with WT osteoblasts. Lack of Sost in osteoblasts further enhanced MALAT1 expression (Figure 3B). Up-regulation of MALAT1 in metastatic prostate cancer (Figure 4A) and PC3 cells co-cultured with SostKO osteoblasts (Figure 3B) suggests that reduced Sost expression in the bone microenvironment may promote prostate cancer metastasis at least in part through the up-regulation of non-coding RNA MALAT1.
Figure 4  LncRNA MALAT1 is transcriptionally modulated by Sost. (A) Heat map showing the expression values of MALAT1 probes in primary prostate cancer samples (primary) and metastatic prostate cancer samples (met). Individual samples are represented as columns and MALAT1 probes as rows; (B) MALAT1 was ~6-fold up-regulated in prostate cancer cells co-cultured with UMR osteoblastic cells and ~5.6-fold down-regulated in PC3 cells treated with recombinant human SOST (rhSOST) compared to prostate cancer monocultures, as confirmed by qPCR.

3.4. Sost Is a Regulator of MALAT1 in Prostate Cancer Cells
Microarray revealed that MALAT1 was up-regulated in prostate cancer cells co-cultured with WT osteoblasts compared to PC3 alone, suggesting that factors secreted by osteoblasts up-regulate MALAT1. Subsequently, we co-cultured PC3 cells with UMR osteoblasts and, using qPCR, confirmed that the factors secreted by osteoblasts up-regulated MALAT1 expression in prostate cancer. The qPCR data showed a ~6-fold increase in MALAT1 expression in PC3 cells co-cultured with UMR osteoblasts compared to PC3 monocultures (Figure 4B). To test whether Sost is a regulator of MALAT1 in prostate cancer we cultured PC3 cells with recombinant human SOST for 48 h and quantified MALAT1 expression using qPCR. Treatment with recombinant SOST resulted in ~5.6-fold reduction in MALAT1 expression (Figure 4B), suggesting that Sost in the tumor microenvironment may have an inhibitory effect on MALAT1 and down-regulation of Sost in the bone microenvironment may enhance MALAT1 expression in prostate cancer.